Cellulose ancestral enzyme based on ancestral sequence reconstruction and uses thereof

Abstract

The present application relates to a kind of persistence endocellulase ancestor and its application.The present application uses the persistence endocellulase of Bacillus subtilis as mother, uses ancestor sequence reconstruction technology, deduces the evolutionary relationship based on systematics analysis, deduces the amino acid sequence of ancestor enzyme from extinct organism using computer calculation.The persistence endocellulase ancestor includes ASR95, ASR106, ASR107, ASR108, ASR109, ASR110, ASR145.Compared with wild-type enzyme, the ancestor enzyme has high catalytic efficiency, good thermal stability and other excellent properties, so that the potential direction of producing reducing sugar by degrading filter paper is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to a class of persistent endoglucanase mutants based on ancestral sequence reconstruction and application thereof. BACKGROUND

[0002] Cellulose is one of the main components of plant cell walls, and is the most abundant organic polymer on earth. The wide source makes cellulose show great potential as a renewable energy source. Some microorganisms can produce extracellular cellulases to decompose cellulose into glucose. However, due to the protection of hemicellulose and lignin in plants, it is difficult to obtain cellulose from plant cell walls, and the process of cellulase degrading cellulose needs to withstand the high temperature and high PH conditions of industrial conversion.

[0003] Processive endoglucanase is a newly discovered bifunctional cellulose hydrolyzing enzyme, which has the characteristics of endoglucanase and the persistent catalytic degradation ability of exoglucanase, and can efficiently degrade cellulose to produce small molecular oligosaccharides. However, the types of processive endoglucanase are few, and the catalytic mechanism of crystalline cellulose is not clear, so it is still a great challenge to improve its catalytic efficiency.

[0004] Although processive endoglucanase has good prospects in cellulose degradation, the wild-type enzyme still needs to be improved in substrate specificity, stability and catalytic efficiency, and industrial application is still a challenge. The relative enzyme activity of wild-type processive endoglucanase derived from Bacillus subtilis is only 40% after incubation at 60℃ for 2h, and it needs to be improved in thermal stability. With the cross development of computer discipline and biology discipline, in the past ten years, ancestral sequence reconstruction technology has been widely used to obtain ancestral enzymes with improved thermal stability.

[0005] Ancestral sequence reconstruction technology successfully reconstructs ancestral sequences by inferring phylogenetic relationships between modern homologous sequences and applying statistical models of amino acids to calculate sequences of internal nodes of phylogenetic trees. The reconstructed ancestral proteins show enhanced thermal stability or mechanical stability, more extreme pH range, improved activity, and substrate promiscuity, in some cases, all at the same time. Changes in these properties are consistent with the hypothesis of extreme environments on Earth during the Precambrian period, such as high temperature and dependence on multiple catalytic functions of the same enzyme system. Therefore, ancestral sequence reconstruction technology is also a technology for improving the stability, substrate promiscuity, and activity of enzymes. For example, Matthew et al. obtained an omega-transaminase that catalyzes more types of substrates than the original enzyme by using ancestral sequence reconstruction technology; Miriam et al. explored the evolutionary process of evolving from an ancestral binding protein lacking isomerase activity to a catalytically active chalcone isomerase by using ancestral sequence reconstruction technology. Philip et al. obtained a serine protease with 14-fold higher activity than the wild-type enzyme by using ancestral sequence reconstruction technology. Nakano et al. obtained an L-arginine oxidase with greatly improved thermal stability by ASR.

[0006] Currently, there is no report on using ancestral sequence reconstruction technology to obtain ancestral enzymes of existing enzymes to improve the thermal stability and activity of persistent endo-cellulase. SUMMARY

[0007] The present application uses ancestral sequence reconstruction technology, based on the analysis of species evolutionary relationships, to obtain a class of persistent endo-cellulase ancestral enzymes with further improved enzyme activity and thermal stability by selecting appropriate amino acid substitution models, thereby providing new feasibility for the biological degradation of cellulose.

[0008] To achieve the above object, the present application adopts the following technical solutions:

[0009] The protein sequence of the persistent endo-cellulase of Bacillus subtilis is input into the uniprot (https: / / www.uniprot.org) website, and homologous sequences are screened to determine the amino acid substitution model using RAXml and construct a phylogenetic tree. PAML is used for ancestral sequence reconstruction, and the protein sequences of each node on the branch of the persistent endo-cellulase of Bacillus subtilis finally differentiated in the phylogenetic tree are selected for cloning and expression. After determination of enzyme activity and thermal stability, a mutant with significantly improved thermal stability is obtained.

[0010] The present application is based on the optimization and modification of persistent endo-cellulase EG5C-1.

[0011] The application provides a persistent endo-cellulase ancestor enzyme based on ancestor sequence reconstruction, which is obtained by mutation of a persistent endo-cellulase from Bacillus subtilis, and the amino acid sequence of the wild-type persistent endo-cellulase is shown as SEQ ID: NO. 1, and the persistent endo-cellulase ancestor enzyme comprises ASR95 (the amino acid sequence is shown as SEQ ID: NO. 2, and the nucleotide sequence is shown as SEQ ID: NO. 9), ASR106 (the amino acid sequence is shown as SEQ ID: NO. 3, and the nucleotide sequence is shown as SEQ ID: NO. 10), ASR107 (the amino acid sequence is shown as SEQ ID: NO. 4, and the nucleotide sequence is shown as SEQ ID: NO. 11), ASR108 (the amino acid sequence is shown as SEQ ID: NO. 5, and the nucleotide sequence is shown as SEQ ID: NO. 12), ASR109 (the amino acid sequence is shown as SEQ ID: NO. 6, and the nucleotide sequence is shown as SEQ ID: NO. 13), ASR110 (the amino acid sequence is shown as SEQ ID: NO. 7, and the nucleotide sequence is shown as SEQ ID: NO. 14), and ASR145 (the amino acid sequence is shown as SEQ ID: NO. 8, and the nucleotide sequence is shown as SEQ ID: NO. 15).

[0012] Another object of the application is to provide application of the persistent endo-cellulase ancestor enzyme in cellulose degradation, and compared with the wild-type enzyme, the mutant enzyme has better thermodynamic stability under high-temperature conditions, and is more suitable for industrial application.

[0013] Another object of the application is to provide an endo-cellulase mutant gene encoding the endo-cellulase mutant, a recombinant vector comprising the endo-cellulase mutant gene and a transformant of the recombinant vector.

[0014] Further, the application also provides a preparation method of the recombinant vector, the endo-cellulase mutant gene is prepared by an artificial synthesis or gene cloning method, an expression vector is constructed to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a host cell.

[0015] The expression vector is a plasmid, a bacteriophage, a virus or a host cell.

[0016] The host cell is a prokaryotic cell or a eukaryotic cell, can be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma, and preferably Escherichia coli.

[0017] The endo-cellulase ancestor enzyme can be used for catalyzing cellulose degradation.

[0018] Compared with the prior art, the present application has the following beneficial effects: the present application adopts ancestor sequence reconstruction, and based on the evolutionary relationship deduced by phylogenetic analysis, uses computer calculation to obtain the endoglucanase mutant with obvious advantages in thermodynamic stability and enzyme activity compared with wild enzyme. Compared with wild enzyme, the activity of the cellulase mutant on substrate Avicel is increased by five times, and after incubation at 60℃ for 2h, the wild type only remains 40% of the activity, while the activity of the mutant enzyme is basically not lost. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 SDS-PAGE electrophoresis analysis of the endoglucanase EG5C-1 and the ancestor enzyme.

[0020] Figure 2 Relative hydrolysis activity of the original enzyme and the ancestor enzyme on CMC and Avicel substrates.

[0021] Figure 3 Temperature stability analysis of the original enzyme and the ancestor enzyme.

[0022] Figure 4 pH stability analysis of the original enzyme and the ancestor enzyme. DETAILED DESCRIPTION

[0023] The specific steps of the present application are illustrated by the following examples, but are not limited by the examples.

[0024] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise specified.

[0025] The present application is further described in detail below by combining specific examples and referring to data. It should be understood that these examples are only for illustrating the present application, and do not limit the scope of the present application in any way.

[0026] In the following examples, various processes and methods not described in detail are conventional methods known in the art.

[0027] Example 1 Ancestor sequence reconstruction of endoglucanase

[0028] The protein sequence of the persistence endo-cellulase EG5C-1 of Bacillus subtilis was uploaded to UniProt (https: / / www.uniprot.org) for blast comparison to obtain 92 protein sequences, raxmlHPC v.8.2.12 was used to select the amino acid model LG to construct a phylogenetic tree, and PAML v4.9 was used to reconstruct the ancestral proteins of endo-cellulase, SEQ ID: NO 2~8. The corresponding ancestral protein gene sequences (SEQ ID: NO 9~15) were obtained by whole gene synthesis, and the corresponding expression strains were constructed (provided by Jinweizhi Biotechnology Co., Ltd.).

[0029] Example 2 Expression of endo-cellulase and ancestral enzyme

[0030] The expression strains of the ancestral enzyme and the original enzyme EG5C-1 constructed above were inoculated into 50 mL of LB liquid medium, respectively, and kanamycin sulfate was added to a final concentration of 100 μg / mL, 180 rpm / min, 37°C overnight culture; the seed liquid of the overnight culture was inoculated into fresh 50 mL of LB liquid medium at a 2% inoculation amount, 180 rpm / min, 37°C constant temperature culture until OD600 was 0.6~1.0, then IPTG (final concentration 0.1 mmol / L) was added, and the expression was induced at 25°C for 24 h.

[0031] The fermentation broth after induction expression was centrifuged at 12000 rpm / min for 20 min, and the supernatant was discarded, then the bacterial body was resuspended with 50 mM Na2HPO4-KH2PO4 (pH 6.0) buffer, then ultrasonic broken, and SDS-PAGE electrophoresis detection was performed, the concentration of the concentrated gel was 4%, the concentration of the separation gel was 12.5%, the sample was mixed with the loading buffer at a ratio of 3:1, and the sample was loaded into the electrophoresis and reacted in a boiling water bath for 5 min. The electrophoresis instrument was set to an initial voltage of 120V, and when the sample moved to the separation gel, the voltage was increased to 230V, and the electrophoresis was ended when the sample moved to the bottom of the electrophoresis tank.

[0032] The results are shown in Figure 1 The molecular weight of the persistence endo-cellulase EG5C-1 was 33.4 kDa, and the results showed that the ancestral enzymes after induction had obvious bands at 33.4 kDa, indicating that the persistence endo-cellulase ancestral enzyme was successfully induced and expressed.

[0033] Example 3 Separation and purification of endo-cellulase and ancestral enzyme

[0034] Since the C-terminal of the persistent endo-cellulase EG5C-1 is fused with six histidine (His) tags, the C-terminal of the mutants also has His tags. The nickel in the nickel column can bind with the protein containing His tags and also can bind with imidazole. Therefore, the nickel column is used to purify the target protein, and the protein elution is achieved by increasing the concentration of imidazole, so as to obtain the purified target protein. The fermentation induced expression of the bacterial liquid is centrifuged and broken, and the obtained supernatant is filtered by a 0.22 μm filter membrane to obtain a crude enzyme liquid, and then a nickel column (GE Healthcare, Fairfield, USA) is used to separate and purify the target protein. The steps are as follows:

[0035] a. The nickel column is flushed with 0.22 μm filtered distilled water at a flow rate of 2 mL / min to wash away the filler protection liquid and compact the filler;

[0036] c. The pretreated protein sample is injected into the sample loop by a syringe at a flow rate of 0.5 mL / min, and the breakthrough peak protein of the sample is collected; d. The nickel column is again flushed with at least 5-10 times the volume of Buffer A (20 mM Tris-HCl, pH 7.5) at a flow rate of 2 mL / min to balance the pH in the nickel column until no protein is eluted;

[0037] e. Gradient elution is used, and each gradient is flushed with at least 5 times the volume of Buffer B (20 mM Tris-HCl, 500 mM imidazole, pH 7.5) at a flow rate of 2 mL / min, and the absorption peak protein of each gradient is collected until no protein is eluted; f. The nickel column is flushed with at least 5-10 times the volume of filtered distilled water at a flow rate of 2 mL / min until the ion curve and protein absorption peak of the purification instrument are displayed;

[0038] g. Add 20% ethanol to store the nickel column;

[0039] h. The desalting column preloaded by GE is used to replace the Buffer containing imidazole with 50 mM Na2HPO4-KH2PO4 (pH 7.0) buffer to remove imidazole in the protein solution;

[0040] i. The collected protein solutions of different gradients are verified by SDS-PAGE to obtain the purified proteins of different ancestral enzymes.

[0041] Example 4 Enzyme activity determination of endo-cellulase and ancestral enzyme

[0042] Enzyme activity unit definition: One enzyme activity unit is defined as the amount of enzyme required to produce 1 mmol of reducing sugar from the substrate per minute at 60°C, pH 6.0.

[0043] Endo-glucanase: Accurately weigh 1 g of sodium carboxymethyl cellulose (CMC-Na) into 100 mL of Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0), mix well, accurately pipette 1.5 mL into a test tube as the enzyme reaction substrate, preheat at 60°C for 5 min, then add 0.5 mL of the appropriately diluted protease solution, place in a 60°C water bath shaker for 10 min, add 3 mL of DNS reagent, boil in a water bath for 5 min, then quickly cool to room temperature. Measure the absorbance value at 540 nm with the inactivated enzyme reaction liquid as the control.

[0044] Exo-glucanase: Accurately weigh 10 g of microcrystalline cellulose (Avicel) into 100 mL of Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0), mix well, accurately pipette 1.5 mL into a test tube as the enzyme reaction substrate, preheat at 60°C for 5 min, then add 0.5 mL of the appropriately diluted protease solution, place in a 60°C water bath shaker for 30 min, centrifuge and pipette the supernatant, add 3 mL of DNS reagent, boil in a water bath for 5 min, then quickly cool to room temperature. Measure the absorbance value at 540 nm with the inactivated enzyme reaction liquid as the control.

[0045]

[0046] Where: X---specific enzyme activity, U / mg;

[0047] 180----------Reducing sugar is converted from milligrams to micromoles;

[0048] Reaction time----------The reaction time of CMC substrate is 10 min, and the reaction time of Avicel is 30 min;

[0049] n-------------Reaction protein content, mg.

[0050] Dilute the purified enzyme solution obtained in Example 2 to a certain concentration and pipette 500 mL into a CMC substrate reaction solution prepared with 1.5 mL of pH 6.0 buffer, react at 60°C for 10 min, then add 3 mL of DNS solution and boil for 5 min after the reaction is completed. Measure the enzyme activity. Take the highest enzyme activity as 100%, calculate the relative enzyme activity in turn, and draw a curve of enzyme activity change with the change of substrate pH.

[0051] The purified enzyme solution was diluted with buffers of different pH, incubated at 4°C for 2h on ice, and then its enzyme activity was determined in the same way as the determination of endocellulase activity. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in sequence to compare the enzyme activity changes between the original enzyme and the ancestral enzyme.

[0052] The results are shown in Figure 1 、 2 The protein expression of ASR95, ASR106, ASR107 and ASR108 was significantly lower than that of the original enzyme. However, compared with the original enzyme, the activity of ASR107 on the substrate Avicel increased by 5 times. The activity of ASR108 on the substrate CMC increased by nearly three times. The enzyme activity of the ancestral proteins at other evolution nodes on the soluble substrate CMC and the insoluble substrate Avicel also increased.

[0053] Example 5 Optimum pH and pH stability of the original enzyme and the ancestral enzyme

[0054] Different pH buffers and substrates were prepared: citric acid-sodium citrate (pH 3.0-6.0), Na2HPO4-KH2PO4 (pH 6.0-8.0), glycine-sodium hydroxide (pH 8.0-9.0), and 1% CMC substrate reaction solution was prepared under different pH conditions.

[0055] The purified enzyme solution obtained in Example 2 was diluted to a certain concentration and 500mL was added to the CMC substrate reaction solution prepared with 1.5mL of different pH buffers. The reaction was carried out at 60°C for 10min, and then 3mL of DNS solution was added and boiled for 5min. The enzyme activity was determined. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in sequence to draw the curve of the change of enzyme activity with the change of substrate pH.

[0056] The purified enzyme solution was diluted with buffers of different pH, incubated at 4°C for 2h on ice, and then its enzyme activity was determined in the same way as the determination of endocellulase activity. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in sequence to draw the curve of the change of enzyme activity with the change of substrate pH.

[0057] The results are shown in Figure 3As shown in Figure 6, the optimum reaction pH of cellulase EG5C-1 and its ancestral enzymes is pH 6.0, and they have the highest hydrolysis activity at pH 6.0. As the pH increases or decreases, the enzyme activity of both the original enzyme and the ancestral enzymes decreases. The stability of the ancestral enzymes is significantly better than that of the original enzyme between pH 6.0 and 9.0, and they can retain more than 90% of the enzyme activity after 2h incubation under various conditions. However, the stability of cellulase EG5C-1 and its ancestral enzymes decreases between pH 3.0 and 5.0.

[0058] Example 6 Optimum reaction temperature and temperature stability of the original enzyme and the ancestral enzymes.

[0059] The purified enzyme solution obtained in Example 2 was diluted to a certain concentration and 500ml was taken into a test tube containing 1.5ml of CMC substrate, and was reacted at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 10min, respectively. After the reaction, 3ml of DNS solution was added and boiled for 5min, and the enzyme activity was measured. Taking the highest enzyme activity as 100%, the relative enzyme activity was calculated in sequence, and the curve of enzyme activity change with temperature was drawn.

[0060] In order to determine the temperature stability of the ancestral enzymes and the original enzyme, the purified enzyme solution was placed in a 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C water bath for 2h, and then the residual enzyme activity was determined according to the method for determining the endo-cellulase activity. Taking the highest enzyme activity as 100%, the relative enzyme activity was calculated in sequence, and the curve of enzyme activity change under different incubation conditions was drawn.

[0061] The results are shown in Figure 7. Figure 4 As shown in Figure 7, the optimum reaction temperature of cellulase and its ancestral enzymes is 60°C. When the temperature is between 30°C and 60°C, the enzyme activity of cellulase and its ancestral enzymes gradually increases. When the temperature is higher than 60°C, the enzyme activity of cellulase and its ancestral enzymes significantly decreases. When the temperature is lower than 50°C, the cellulase and its ancestral proteins still retain more than 80% of the enzyme activity after 2h incubation. When the temperature is higher than 60°C, the cellulase EG5C-1 retains 30% of the activity, and ASR95, ASR106, ASR107, ASR108, and ASR109 retain more than 80% of the activity. ASR108 and ASR106 almost have no loss of enzyme activity.

Claims

1. An endocellulase ancestor enzyme characterized in that, The amino acid sequence is shown as SEQ ID NO:

4.

2. A gene encoding the endocellulase ancestor enzyme according to claim 1.

3. A recombinant expression vector comprising the gene according to claim 2.

4. The recombinant expression vector of claim 3, wherein, The recombinant expression vector is a plasmid, a virus.

5. A host cell comprising the gene of claim 2, characterized in that, The host cell is selected from the group consisting of Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

6. The host cell of claim 5, wherein, The host cell is Escherichia coli.

7. A method of producing the endocellulase ancestor enzyme of claim 1, wherein, The host cell is transformed with the recombinant expression vector according to claim 3 to obtain a recombinant strain, and the endocellulase ancestor enzyme is induced to express.

8. Use of the endocellulase ancestor enzyme according to claim 1 in cellulose biodegradation.

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